IL-6, IL-11, ciliary neurotropic factors, leukemia inhibitory factors, oncostatin-M, and cardiotropin-1 belonging to IL-6 type cytokines are known to transmit signals through a receptor complex containing at least one signal-transmitting protein gp130. For example, IL-6 links to IL-6R, and the resulting IL-6•IL-6R complex contains gp130.
The hematopoiesis system is one of the living body protection systems in which IL-6 plays an important role. The process for expression of IL-6R in hematopoietic cells is different between human cells and mouse cells. Undifferentiated human hematopoietic precursor cells which can form a granulocyte macrophage colony, an erythroblast colony, a megakaryocyte colony, and a mixed colony thereof on a methylcellulose culture do not express a sufficient amount of IL-6R (Tajima et al.: J. Exp. Med. 184, 1996). Therefore, the undifferentiated human hematopoietic precursor cell is almost unreactive to IL-6, whereas it is strongly reactive to an IL-6•IL-6R complex. In contrast, the undifferentiated mouse hematopoietic precursor cell expresses a sufficient amount of IL-6R. Nakahata, and the inventors of the present invention found that the human megakaryocte precursor cell does not express a sufficient amount of IL-6 (Japanese Patent Application No. 9-325847). These findings are consistent with the fact that IL-6 administered to a mouse increases significantly the number of blood platelets and the IL-6 administered to a human body is limited in its effect.
The linking constant between IL-6 and soluble IL-6R is reported to be 5×10−9 M (Yasukawa et al.: J. Biochem. vol. 108, p. 673, 1990). This means, that in a mixture of 200 ng/mL (1×10−8 M) of IL-6 (mw: 20000) with 500 ng/mL (1×10−8 M) of soluble IL-6R (mw: 50000), half of the molecules will exist in an unlinked state. Actually, soluble IL-6R is required in an amount of 1000 ng/mL or more to react it with cells not expressing IL-6R.
By genetic engineering, two kinds of proteins existing independently in nature can be fused into a fusion protein of one polypeptide chain. When two kinds of proteins linkable with each other are expressed as a fusion protein, presumably the bonding is strong and dissociation is less liable to occur as long as the respective proteins in the fused state can keep the inherent structures (biologically active structure)
For two kinds of proteins in a fused protein to take the inherent structures, steric hindrance should not be caused between the two proteins. Further, the two proteins in the fused protein having the inherent structures should have freedom degrees for contacting each other. Therefore, a linker is employed conventionally for fusion of the proteins: the linker being a sequence of 5-20 amino acid residues such as a glycine residue and a serine residue having a high freedom degree for linking of two proteins. By such indirect fusion of proteins through a linker not related to the fused proteins, the steric hindrance between the two proteins is avoided and the freedom degree for the linking is achieved.
For example, for expression of a fusion protein by bonding a V domain of an H chain with a V domain of an L chain of antibodies affinitive for bonding, a linker sequence is disclosed which has a residue sequence of GGGGSGGGGSGGGGS (G: glycine residue, S: serine residue) (SEQ ID NO:61) (Houston et al.: Proc. Natl. Acad. Sci. USA, 85, p. 5879, 1988).
Fusion proteins were prepared recently from IL-6 and IL-6R linkable to each other with a linker (illustrated in FIG. 1(a)). One is a fusion protein prepared in a manner such that an irrelevant linker RGGGGSGGGGSVE (SEQ ID NO:60) not contained in IL-6 and IL-6R is bonded to the C-terminal 323th alanine residue in IL-6R, and IL-6 is bonded to its C terminal side (Fisher et al.: Nature Biotech, 15, p. 142, 1997). A second one is a fusion protein prepared in a manner such that a linker EFM (E: glutamic acid residue, F: phenylalanine residue, and M: methionine residue) is bonded to the C-terminal 356th valine residue of IL-6R, and further IL-6 is bonded to its C terminal side (Chebath et al.: Eur. Cytokine Netw, 8, p. 359, 1997). The inventors of the present invention discovered a fusion protein prepared by bonding a linker SSELV (L: leucine residue, V: valine residue) (SEQ ID NO:62) to the C-terminal 334th leucine residue of IL-6R, and further bonding IL-6 to the C-terminal thereof (Japanese patent Application No. 10-2921).
Generally, it is known that a foreign protein expressed by genetic recombination can be cleaved by a naturally expressed protease in the host. Therefore, the fusion protein IL-6R•IL-6 having expressed may also be cleaved by protease in the host. In particular, the yeast Pichia pastoris is known to express various proteases. However, the aforementioned reports do not describe this matter. If the fusion protein IL-6R•IL-6 is cleaved by a protease expressed in the host, preparation of a fusion protein IL-6R•IL-6 resistant to the cleavage enables production of a larger amount of the fusion protein IL-6R•IL-6 from the culture of the yeast Pichia pastoris or other host.
A fusion protein like IL-6R•IL-6, which can maintains firmly the fused state of two proteins, is expected to be particularly effective in a fused state as a medicine in signal transmission system of IL-6 in a human body or a like animal.
In development of a fusion protein as a periodically dosed medicine, high possibility of the aforementioned immune reaction is a problem, since the linker is not contained in the proteins and irrelevant to them, and has an independent steric structure. Therefore, the linker has preferably a sequence as short as possible, or nonuse of the linker is desirable.
As described above, for constituting the fusion protein, the two constituting proteins should not cause steric hindrance between them, and should have freedom for linking together. For example, for direct fusion of IL-6R and IL-6 without using a linker without causing steric hindrance with a freedom degree for linking together, many factors should be decided such as the order of the proteins in fusion, the amino acid residue in the N-terminal side protein and the that in the C-terminal side protein.
As the fusion protein of IL-6R with IL-6, only three examples mentioned above have been reported which employ a linker, and the fusion necessarily requires a linker. FIG. 1(a) illustrates a fusion protein in which two proteins are linked together through a linker. The direct linking without the linker has not been reported yet.
The present invention intends to provide a IL-6R•IL-6 fusion protein in which IL-6R and IL-6 are linked directly without a linker as illustrated in FIG. 1(b). The present invention intends also to provide an IL-6R•IL-6 fusion protein which is resistant to cleaving action of the protease expressed by a host, especially a Pichia pastoris type yeast, and a gene for coding for the fusion protein.